Mechanisms of CCD-induced activation of vagal afferent neurons

Cholecystokinin (CCK), a gastrointestinal hormone released from endocrine I cells in response to the entry of macronutrients, suppresses food intake by promoting satiation via a vagally mediated mechanism. In vitro, CCK can directly activate cultured vagal afferent neurons by modulating complementary background conductances. However, the identity of these conductances, as well as the transduction pathway involved in this process, has not been identified. To address theses questions, we first determined the presence of messenger RNA for several TRP and K2P channels, which are promising candidates for the background conductances modified by CCK. Indeed, we found that TRPC1/3/5/6, TRPV1-4, TRPM8, TRPA1, TWIK2, TRAAK, TREK1, and TASK1-2 were all present in rat nodose ganglia. We then used quantitative RT-PCR, single-cell PCR, combined with systemic capsaicin lesion and retrograde labeling techniques, to investigate the distribution of selected TRP and K2P subtypes. Our results demonstrate that these background conductances are indeed differentially distributed in the nodose, and they not only segregate with specific markers such as TRPV1 or CCK-1 receptors, but the degree of overlap is also dependent on the innervation target. However, none of the specific conductances was segregated with CCK-1 receptors at a frequency that would suggest they are obligatory for signaling by CCK-1 receptors. We next investigated the ion conductances that are involved in the CCK-induced activation using calcium imaging and electrophysiological approaches. By use of various reagents that interact with different TRP subtypes or other ion channels, we demonstrated that CCK-induced calcium signals are not secondary to the depolarization provoked by CCK, and that the activation induced by CCK is likely to be mediated by more than one members of the TRPV family, excluding TRPV1. Our data also suggest that additional ion subtypes may participate under certain circumstances, but further investigations are needed to test that possibility. Finally, we assessed the roles of various pathways in the calcium responses to CCK in nodose neurons using activators and inhibitors of specific pathways. We concluded that CCK-elicited calcium signals are likely to involve PLC, but do not require PKC, PLA2, PKA, or PI3K, or release from the intracellular store